The present invention relates to a high beta-conglycinin composition, meat analog, cheese analog, beverage and animal feed and to methods of producing a high beta-conglycinin composition, cheese analog, beverage, meat analog and animal feed.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference.
Glycinin and beta-conglycinin (BC) account for approximately 70% of the proteins in soybeans. It has been postulated that the functional properties of soy protein ingredients in food systems can be improved by modifying the ratio of these proteins. Previous attempts have been to increase the ratio of glycinin to beta-conglycinin to improve the yield and quality of tofu-type soybean gels and to improve the content of sulfur amino acids for nutritional purposes (Kitamura, K., Trends Food Science & Technology 4:64-67, (1993), Murphy, P., et al., Food Technology 51:86-88, 110 (1997)).
Dietary proteins are needed to replace metabolic losses of tissue and organ proteins, to form and deposit protein in new tissues and to replenish tissue loss as a consequence of pathological conditions. These needs are met by indispensable (essential) amino acids and dispensable amino acids that comprise dietary proteins. It is largely in this context that the nutritional value of dietary proteins is defined as the ability to meet daily requirements for essential amino acids (Steinke, F. et al. New Protein Foods in Human Health: Nutrition Prevention and Therapy, CRC Press, 1992). High quality proteins contain all the essential amino acids at levels greater than reference levels and are highly digestible so that the amino acids are available. In this context, egg white and milk proteins are the standards to which other proteins are evaluated and plant proteins are considered to have inferior nutritional value. The essential amino acids whose concentrations in a protein are below the levels of a reference protein are termed limiting amino acids, e.g., the sum of cysteine and methionine are limiting in soybeans.
Glycinin contains 3 to 4 times more cysteine and methionine per unit protein than beta-conglycinin (Fukushima D., Food Rev. Int. 7:323-351, 1991). Thus it is expected that an increase in the content of glycinin and a decrease in the content of beta-conglycinin results in enhanced protein quality (Kitamura, K. Trends Food Science & Technology 4:64-67, 1993; Kitamura, K., JARQ 29:1-8, 1995). This is consistent with the finding that the mean value of the sulfur-containing amino acid contents in the seeds of four representative lines which were low in beta-conglycinin was about 20% higher than that of four ordinary varieties (Ogawa, T. Japan. J. Breed. 39:137-147, 1989). A positive correlation was also reported between the glycinin:beta-conglycinin ratio (1.7-4.9) and the methionine or cysteine concentration of total protein, among wild soybeans (Kwanyuen et al., JAOCS 74:983-987, 1997). There are no reports of the amino acid composition of high beta-conglycinin soybeans (glycinin:beta-conglycinin ratio less than 0.25).
In addition to the ability of proteins to meet the body's daily needs for essential amino acids, dietary proteins can also contribute bioactive peptides and amino acid patterns which can reduce the risk factors for cardiovascular diseases, cancer and osteoporosis. These compositional factors should also be considered in assessing protein quality, especially in countries such as the United States where people on the average consume a large excess of dietary protein. Researchers (Sugano, et al. PCT No. WO89/01495; Sugano, M. J. Nutr 120:977-985, 1990; Sugano. M. & Kobak, K. Annu. NY Acad. Sci. 676:215-222, 1993; Wang, M. J Nutr. Sci. Vitaminol. 41:187-195, 1995) have identified a pepsin-resistant fraction of soybean protein (5,000-10,000 molecular weight) that represents about 15% of the protein in isolated soy protein. Humans fed a diet with the pepsin-resistant fraction at 24 g or 48 g per day had lower LDL-Cholesterol and more fecal neutral and acidic steroid excretion than those fed diets with isolated soy protein or casein. The soy proteins which contribute to this pepsin-resistant fraction were not identified. Purified beta-conglycinin is more pepsin-resistant than purified glycinin (Astwood, J. & Fuchs, R. In Monographs in Allergy, Sixth International Symposium on Immunological and Clinical Problems of Food Allergy, Ortolani, C. and Wuthrich, B. editors, Basel, Karger, 1996), so it follows logically that beta-conglycinin may be a primary contributor to the bioactive fraction. This possibility has not been demonstrated yet in a feeding study, or with protein made from soybeans having altered protein compositions.
The alpha and alpha-prime subunits of beta-conglycinin specifically interacted with membrane components of human and animal liver cells in tissue culture experiments (Lovati, M. R., et al., J. Nutr. 126:2831-2842). The beta-conglycinin subunits were incorporated by the liver cells, degraded and caused an increase in the maximal binding of LDL to high-affinity receptors. It is proposed that such a mechanism could be responsible for the cholesterol lowering properties of soy protein isolates. However, it is not clear if significant amounts of dietary soy proteins can get to the liver in vivo. Lavarti et al. (J. Nutr. 122:1971-1978, 1992) reported a study in which hypercholesterolemic rats were fed either glycinin or beta-conglycinin for two weeks. Both groups showed a ⅓ reduction in total serum cholesterol. There are no studies which determine the effects of soy protein isolates from soybeans with modified soy protein compositions on the cholesterol lowering properties of soy protein isolate in animal models or humans.
It is reasoned from Rhesus monkey studies using alcohol extracted (which removes isoflavones) and non-alcohol extracted soy protein isolate, that soybean isoflavones are the primary components of soy protein isolates responsible for the cholesterol lowering effects (Anthony, M. S., J. Nutr. 126:43-50, 1996). However, subjecting soy protein to ethanol extraction did not have any effect on its lipid-lowering effects in other studies using hamsters (Balmir et al., J. Nutr. 126:3046-3053, 1996) or rats (Topping et al., Nutr. Res. 22:513-520, 1980). Alcohol extraction processes can extract some proteins and can denature and aggregate the unique structures of soy proteins, likely affecting how they act in the GI tract. For example, Sugano et al., (J. Nutr. 120:977-985, 1990) observed that methanol extraction completely eliminated the ability of high molecular weight soy protein peptides to bind and excrete steroids. Feeding isolated soy isoflavones (genistein and daidzein) had no Favorable effect on serum lipids or lipoproteins in humans (Colquhoun, et al., Atherosclerosis, 109:75, 1994; Nestel, P. J., Arterioscler. Thromb. Vasc. Biol. 17:3392-3398, 1997).
The confusion about the relative roles of various soy protein isolate constituents in the observed cholesterol-lowering effects, are difficult to resolve by using processing technologies to create samples with altered composition. An improved approach is to specifically modify the components of interest in the soybeans.
An emerging key indicator for the risk of heart disease, is high serum homocysteine levels. Dietary methionine is a precursor to homocysteine, so a high consumption of methionine can potentially increase consumers risk of heart disease, especially if they also consume low levels of folic acid and vitamin B6 (McCully, K. S., The Homocysteine Revolution, Keats Publishing, Inc., New Canaan, Conn., 1997). Another route which lowers the endothelial cytotoxicity of homocysteine is the reaction between nitric oxide (NO) and homocysteine in vivo to form the non-toxic S-nitroso-homocysteine. This route can be enhanced by increasing dietary arginine levels because arginine is converted by nitric oxide synthase to NO. Therefore, an ideal dietary protein for maintaining healthy levels of homocysteine, should have high arginine and low methionine (and cysteine), as is found in beta-conglycinin. However, the use of a beta-conglycinin rich soy protein isolate designed for this purpose has not been previously disclosed.
New protein ingredients must contribute positively to the taste, texture and appearance of foods to gain acceptance. These quality attributes are determined by the structure of the proteins and how they change in the presence of other food components (e.g., calcium ions, other proteins) and processing conditions (e.g., temperature, pH). Increasing the glycinin content of soybeans is usually proposed for improving food functionality of soy protein ingredients. Previous attempts to improve the yield and quality of tofu-type soybean gels have been to increase certain glycinins or the ratio of glycinin to beta-conglycinin (Wang, C-C. and Chang, S. J. Agric. Food Chem. 43:3029-3034, 1995; Yagasaki, K. et al. Breeding Sci. 46:11-15, 1996; Murphy, P., et al., Food Technology 51:86-88, 110, 1997). There is little information on the properties of glycinin and beta-conglycinin in other model food systems, especially under conditions typical of other food systems (e.g., low pH, high salt, fat, gel formation at temperatures below 72 degrees C.). Foaming properties of glycinin are superior to those of beta-conglycinin at a pH of 7.0 and no salt (Yu, M. A., J. Agric. Food chem. 39;1563-1567, 1991). Partially hydrolyzed glycinin forms heat-induced gels which are more similar to cheese curd than partially hydrolyzed beta-conglycinin at neutral pH (Kamata et al., Nippon Shokuhin kogyo Gakkaishi 36:557-562, 1989). Glycinin forms gels at boiling temperature with higher elastic moduli than soy protein isolate (Van Kleef, Biopolymers 25:31-59, 1986). Some comparisons were made between glycinin and beta-conglycinin at pH 7.5-8.0 (Shimada, K. and Matsushita, S., Agric. Biol. Chem. 44:637-641, 1980; Utsumi, S. and Kinsella, J. Food Sci. 50:1278-1282, 1985; Nakamura et al., Agric. Biol. Chem. 50:2429-2435, 1986). Though beta-conglycinin was observed to have superior emulsifying properties compared to glycinin, it did not have better emulsifying properties compared to whole soy protein isolate controls (Aoki et al., J. Food Sci. 45:534-546, 1980; Yao et al. JAOCS 67:974-979, 1990). The freeze-thaw properties of beta-conglycinin and glycinin rich soy protein isolates have not been reported, though the problem of soy protein freeze-thaw instability is known (Abtahi, S. and Aminlari, M., J. Agric. Food Chem. 45:4768-4772, 1997).
Soybean germplasm which lack glycinin, sib-line varieties B2W(2), B2G(2), and B2G(1) were received from Dr. Norihiko Kaizuma, President of Tohoku University, Morioka, Japan (Oct. 17, 1996). The mutation of these soybean lines was induced by using gama-irradiation (Odanaka, H. and N. Kaizuma, Japan J. Breed. 39 (Suppl.) 430-431, 1989; Kaizuma et al. Jpn J. breed. 40 (Supple 1) 504-505, 1990). These lines lack all of the group-I subunits consisting of A1aB2, A1bB1b and A2B1a. Synthesis of the missing polypeptides has been shown to be controlled by a single recessive allele. No deleterious effects on physiological aspects such as seed development and germination were observed.
The properties of high beta-conglycinin isolates at pH 7 were discussed in Nagano, T. J. Agric. Food Chem. 44:3484-3488. The gel-forming properties at 85 degrees C. and foaming properties of enzymatically hydrolyzed beta-conglycinin fractions were discussed in Lehnhardt, W. F. and Orthoefer, F. T., European Patent No. 0072617, 1982. The concept of altering seed storage proteins by transgenic methods was made by Kinney, A., et al., International Publication No. WO 97/47731, however only attempts at eliminating beta-conglycinins were made and demonstrated.
Yields of protein and other soybean constituents also need to be considered in designing a commercially viable variety. Positive correlations were found between total protein content of soybeans and the glycinin:beta-conglycinin ratio, so the soybeans that were richer in glycinin had a higher protein content (Shui-Ho Cheng, 1984 Ph.D. thesis, Univ. of Ill.).